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Three-dimensional tracking of a motile microorganism


The microorganism tracking system was developed to track a freely swimming microorganism two-dimensionally. This system, however, could not track the target movement in depth direction.

To solve this problem, dynamic focusing using the depth-from-diffraction (DFDi) method was applied to three-dimensional tracking of a swimming paramecium.

Experimental system was developed to demonstrate the three-dimensional tracking of a paramecium. The system was consist of an optical microscope, an XYZ automated stage, a high-speed vision system (I-CPV), and a CCD camera for monitoring. It can track the specimen three-dimensionally by controlling the chamber position using X and Y axis of the stage to keep the specimen within the field of view, and by controlling the depth position of the chamber to keep the specimen focused using DFDi method.

Three-dimensional tracking of a swimming paramecium for 70 s was successfully demonstrated. Right figure shows a sequence of images captured by the monitoring CCD camera while the paramecium turned from facing the bottom of the chamber to facing the top, while tracking the paramecium to keep it on the focal plane. The focal position variance can be seen by observing a dust particle indicated by the circles A, B, and C in the figure. The specimen's trajectory can be calculated from the sequence of the XYZ stage position.

System diagram of the three-dimensional tracking system and photographs of the tracked paramecium.


Computer generated animation of the tracking experiment

How the system tracked a swimming paramecium


  1. Hiromasa Oku, Masatoshi Ishikawa, Theodorus, and Koichi Hashimoto, "High-speed autofocusing of a cell using diffraction pattern," Optics Express 14, 3952-3960 (2006).
  2. Hiromasa Oku, Theodorus, Koichi Hashimoto and Masatoshi Ishikawa. High-speed Focusing of Cells Using Depth-From-Diffraction Method. 2006 IEEE International Conference on Robotics and Automation (ICRA 2006) (Orlando, U.S.A., 2006.5.18) / Proceedings, pp. 2626-2641. [PDF (716K)] *IEEE
  3. Theodorus, Hiromasa Oku, Koichi Hashimoto and Masatoshi Ishikawa. Optical axis tracking of microorganism using highspeed vision. Focus on Microscopy 2005 (Jena, Germany, 2005.3.22) / Program and Abstract Book, pp. 105.

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